A confluence of different areas of immunological research offers an opportunity for a more synthetic approach to investigate T cell recognition of antigens in patients with MS, an inflammatory disease of the CNS myelin. These include the elucidation of the crystal structure for TCR recognition of antigen/MHC; identification of potential biochemical techniques that provide insights into the cell biology of class II restricted presentation with particular emphasis on the proteases involved in this process. In the first project, we shall utilize the newly developed MHC tetramer technology to determine the frequence, activation state and cytokine profile of antigen specific T cells in the CSF & blood of HLA- DR2 MS patients, which has almost certainly been underestimated by limiting dilution techniques. The presentation of myelin peptides by APC's in MS lesions will be investigated using TCR tetramers generated with cDNAs from myelin specific T cell clones and used to stain MS plaque tissue. In the second project, combinatorial peptide libraries will be used to examine the antigen specificity of oligoclonal CSF T cells. These investigations will also be performed on subjects with defined CNS infections and will determine whether TCRs expressed on CNS T cells in patients with MS are degenerate in their recognition of combinatorial peptide libraries as compared to CNS T cells in patients with MS are degenerate in their recognition of combinatorial peptide libraries as compared to CNS T cells recognizing viral or bacterial antigens. It will be determined whether oligoclonal T cells and MBP/DR2 tetramer binding T cells are reflective of the original CSF population Project 2 will explore the cell-biological and biochemical properties of CNS microglial cells. The processing pathway of the myelin autoantigens MBP and MOG and the trafficking of Class II molecules and MOG in human microglia will be examined. Preparations of MBP and MOG obtained by in vitro transcription/translation will serve as a source of native, radiolabeled antigen, the processing of which will be monitored using biochemical techniques. These experiments will examine the role of lysosomal proteases Cathepsin B, D, L or S, likely candidates for the proteolytic conversion of MBO and MOG in antigen processing. MOG and MBP visualization in situ of antigen-bearing APCs in project 1 will be used to examine the processing of these antigens. The visualization in situ of antigen-bearing APCs in project 1 will be used to examine CNS microglial cells isolated in project 3. Analysis of antigen-specific CD4+ T cells with sensitive techniques will have important implications for understanding the pathogenesis of MS and for immune monitoring by any form of therapy.